Activity-based protein profiling of human and plasmodium serine hydrolases and interrogation of potential antimalarial targets

Summary Malaria remains a global health issue requiring the identification of novel therapeutic targets to combat drug resistance. Metabolic serine hydrolases are druggable enzymes playing essential roles in lipid metabolism. However, very few have been investigated in malaria-causing parasites. Here, we used fluorophosphonate broad-spectrum activity-based probes and quantitative chemical proteomics to annotate and profile the activity of more than half of predicted serine hydrolases in P. falciparum across the erythrocytic cycle. Using conditional genetics, we demonstrate that the activities of four serine hydrolases, previously annotated as essential (or important) in genetic screens, are actually dispensable for parasite replication. Of importance, we also identified eight human serine hydrolases that are specifically activated at different developmental stages. Chemical inhibition of two of them blocks parasite replication. This strongly suggests that parasites co-opt the activity of host enzymes and that this opens a new drug development strategy against which the parasites are less likely to develop resistance.


INTRODUCTION
Despite a significant drop in incidence over the last two decades, malaria remains a global health problem with 229 million estimated cases and close to half a million deaths in 2019 (WHO, 2020). Malaria is caused by parasites of the Plasmodium genus and is transmitted by Anopheles mosquitoes. Plasmodium falciparum is the most virulent species responsible for 90% of mortality. Malaria control has been hampered by the rise of insecticide-resistant mosquitoes and drug-resistant parasites (WHO, 2018). It is therefore imperative to develop new therapies to treat malaria. To slow down the emergence of resistance, new drug candidates should have a novel mechanism of action (Burrows et al., 2013(Burrows et al., , 2017. Plasmodium parasites have a complex life cycle that involves sexual replication in the mosquito host and asexual replication in humans. In the human blood stream, parasites replicate exponentially inside red blood cells (RBCs). This asexual erythrocytic cycle is responsible for the vast majority of malaria pathology. Therefore, new antimalarial drugs need to be highly effective at this stage, but an ideal treatment should also kill other developmental stages to block and prevent transmission.
Serine hydrolases (SHs) are a large family of druggable targets that remain largely unexplored in Plasmodium species. The SH superfamily includes all enzymes that use a nucleophilic serine to catalyze the hydrolysis of bonds, such as amide, ester, or thioester groups. These reactions proceed by a conserved catalytic mechanism, i.e., formation of an acyl-enzyme intermediate that undergoes water-induced hydrolysis to release the products. We can take advantage of this common mechanism to design broadspectrum irreversible inhibitors and probes (Bachovchin and Cravatt, 2012). SHs are involved in many biological processes such as metabolism, post-translational modification, and proteolysis. Metabolic SHs are ubiquitous in all organisms and important in almost all human diseases (Long and Cravatt, 2011); for example, acetylcholinesterase in neurotransmission (Lane et al., 2006), phospholipase A2 in inflammation (Bonventre et al., 1997), and lipases in bacterial infection (Shao et al., 2007). Serine proteases have been extensively studied in malaria and pursued as drug targets (Deu, 2017) but only a few metabolic SHs have been characterized: Pro-drug resistance esterase PARE (Istvan et al., 2017), Bud One common biological function of SHs is in lipid metabolism and membrane biogenesis. RBCs perform very little de novo lipid biosynthesis, therefore P. falciparum parasites must synthesize and scavenge their own phospholipids (Asahi et al., 2005;Dé champs et al., 2010aDé champs et al., , 2010bMarks et al., 1960). Membrane dynamics are extremely important as the parasite replicates and undergoes morphological changes during the erythrocytic cycle, which can be divided into four stages: ring, trophozoite, schizont, and merozoite ( Figure 1A). Invasion of RBCs by merozoites results in invagination of the RBC membrane and formation of the parasitophorous vacuole (PV), within which the parasite develops and replicates (Paul et al., 2015, Riglar et al., 2011. In ring stage (0-20 h post invasion, hpi) the host cell is remodeled by the secretion of parasite proteins into the RBC cytosol and membrane (Zuccala and Baum, 2011). This puts in place nutrient acquisition pathways and strategies to evade the host immune system that require the formation of large membrane structures such as Maurer's Clefts and the tubovesicular network (Sherling and van Ooij, 2016). Trophozoite stage (20-36 hpi) is a very metabolically active period characterized by hemoglobin digestion in the food vacuole, a pathway that liberates space within the RBC and provides a source of amino acids for protein synthesis (Elliott et al., 2008). Large vesicles known as cytostomes transport hemoglobin from the RBC cytosol into the food vacuole. The schizont stage (36-48 hpi) consists of asynchronous nuclear division, membrane biogenesis, and finally, cytokinesis to form 20-32 daughter merozoites (Bannister and Mitchell, 1989). The final step in the life cycle is merozoite egress from the infected RBC (iRBC), a process that is tightly regulated by kinases such as cGMP-dependent protein kinase PKG and proteases such as subtilisin-like protease 1 (SUB1, Blackman, 2008;Collins et al., 2013b). Eighty percent of genes expressed in this cycle do so in a cyclic manner (Bozdech et al., 2003). Given the changing metabolic requirements and membrane remodeling/biogenesis throughout the cycle, PfSHs likely perform essential functions at different stages and require temporal expression and/or activation. The antiplasmodial natural product salinipostin A targets multiple PfSHs and arrests parasite growth in schizont stage (Yoo et al., 2020).
The aims of this study are to profile active SHs throughout the erythrocytic cycle of P. falciparum using quantitative chemical proteomics and to evaluate some of the identified SHs as potential antimalarial targets. Activity-based probes (ABPs) specifically and covalently react with the key catalytic nucleophilic residue of an enzyme via an electrophilic warhead. Fluorophore and affinity tags within the probe can be used to visualize or pull-down modified targets. Because the warhead only reacts with the catalytic residue in a functional active site, ABPs discriminate between active and inactive forms of an enzyme. In activity-based protein profiling (ABPP), broad-spectrum ABPs are used to profile the activity of a whole enzyme family by taking advantage of its conserved catalytic mechanism. ABPP can be used to look at changes in enzyme activation during a biological process such as cell fate commitment, life cycle progression, or in response to external stimuli (Cravatt et al., 2008;Liu et al., 1999;Niphakis and Cravatt, 2014;Willems et al., 2014).  (Schwach et al., 2015). b (Zhang et al., 2018). c (Aurrecoechea et al., 2009;Florens et al., 2002;Lasonder et al., 2015;Oehring et al., 2012;Silvestrini et al., 2010). d (Aurrecoechea et al., 2009;Gó mez-Díaz et al., 2017;Ló pez-Barragá n et al., 2011;Otto et al., 2010;Pelle et al., 2015;Toenhake et al., 2018;Zanghì et al., 2018). e (Istvan et al., 2017). f Identified in FP-Biotin schizont screen (Elahi et al., 2019b). G, gametocyte; ES, early schizonts; M, merozoites; Oc, oocyst ;Ok, ookinetes; R, rings; S, schizont; Sp, sporozoites; T, trophozoites. g (Burda et al., 2015). h Dr Natalie Spillman, personal communication.
i (Spillman et al., 2016). j Dr S. Ridewood, personal communication. k (Groat-Carmona et al., 2015). l (Mailu et al., 2015). iScience Article (48 hpi, pre-egress) schizont stages were treated with 1 mM FP-N 3 or DMSO for 1h. Intact cells were then washed to remove excess unreacted probe before saponin treatment. Parasite pellets were washed multiple times with PBS to remove excess hemoglobin and frozen in liquid N 2 . Merozoites (48 hpi, post-egress) have a very short lifespan outside the RBC and are difficult to collect in large quantities. Therefore, they were collected and frozen before probe (1 mM FP-N 3 ) or DMSO treatment in lysates for 1h. Ring, trophozoite and early or late schizont stage parasites were treated with 1 mM FP-N 3 or DMSO under intact conditions. Parasites were then washed, saponin permeabilized and frozen. Merozoites were lysed before probe labeling. The soluble protein was then extracted, and CuAAC-chemistry used to attach biotin to labeled proteins for pull-down on neutravidin agarose beads. After on-bead reduction and alkylation of proteins, trypsin digestion liberates peptides for subsequent processing for proteomics. Peptide samples are labeled with different 10-plex TMT tags and combined before high pH fractionation and LC-MS/MS analysis. See also Figure S1. iScience Article For each life stage and treatment, triton X-soluble protein fractions were extracted and pooled. Samples of equal protein content were split into three technical replicates and treated with CuAAC-chemistry reagents to conjugate an alkyne-biotin tag to FP-N 3 modified proteins. Labeled proteins were affinity purified using a 10:20 mL mixture of neutravidin:blank agarose beads. These conditions were chosen to optimize depth of coverage ( Figure S1). Captured proteins were reduced, alkylated and trypsin digested on-bead, and the eluted peptides cleaned up by stage tipping. Peptides obtained from each of the ten different conditions (five life stages treated with FP-N 3 or DMSO) were labeled with different TMT tags and combined. Each of the triplicate 10-plex TMT mixes was split into 8 fractions by high-pH fractionation before LC-MS/MS analysis to decrease sample complexity and increase depth of coverage.  Table S1): 27 P. falciparum proteins and 8 HsSHs that are discussed below.
Of the 27 enriched P. falciparum proteins, only two are not predicted to be SHs: a proteasome proteolytic subunit (beta type-5) and an endonuclease (MUS81). Proteasome subunits have been previously identified with FP probes (Jessani et al., 2005), but the interaction with MUS81 is unknown. Of the 25 PfSHs identified, three were PLPs and the rest Pfa/bHs including predicted lysophospholipases, lipases, and peptidases (Table 1). We were surprised to detect PfXL2, a non-essential exported lipase, which we expected to be lost after saponin treatment (Spillman et al., 2016). However, the related PfXL1 was not detected. Among the 25 PfSHs identified here, eight were unique to this study compared to the previous one where they use the FP-desthiobiotin probe in schizonts lysates (Elahi et al., 2019b). Two of these, PF3D7_1476700 (Psta1) and PF3D7_1427100, demonstrate the highest activity in merozoite and trophozoite stages respectively, which might explain their absence from the FP-Biotin study. The remaining six are active in schizont stage but may be less active in lysates or might not have been detected because of differences in sample preparation. On the other hand, the previous study identified three unique PfSHs (Elahi et al., 2019b): PF3D7_1126600, predicted to be a steryl ester hydrolase; PfEH2, which is associated with the RBC periphery (Spillman et al., 2016); and the rhomboid protease ROM4, involved in the shedding of merozoite surface proteins during RBC invasion. These three SHs were detected in the insoluble fraction of schizont lysates and might be strongly associated with membranes. Indeed, ROM4 has seven transmembrane domains. Therefore, our lysis/solubilization conditions might not have been strong enough to extract these SHs.
To quantify changes in SHs activities during the asexual cycle, the average log 2 enrichment at each life stage was used to generate a heat map of hydrolase activity over time ( Figure 3A). SHs were grouped by Euclidean clustering based on their temporal profile. The majority of PfSHs had 4-to 64-fold changes in activity between life stages, with the lowest level of activity usually at ring stage. The dynamic temporal activation of PfSHs suggests they may play stage-specific roles in metabolism. PF3D7_1129300, PF3D7_1476800, PF3D7_0629300 (PfLCAT) and PF3D7_1134500 seem to be most active at schizont stages, and PF3D7_1458300, PF3D7_1358000 and PF3D7_1120400 (abH112) in early schizonts. The activities of Psta1, PF3D7_1328500 (MLPL), and RhoSH increase significantly between mature schizonts and merozoites. We hypothesized that changes in activity between these two stages may represent an activation event essential to egress or invasion.

Genetic interrogation of PfSHs by conditional allelic replacement
Four SHs of unknown function were selected for genetic interrogation. Of the SHs active at merozoite stage, MLPL was annotated as dispensable in a genome-wide piggyBac transposon screen (Zhang et al., 2018) and was not pursued in this study. RhoSH was annotated as important and was already under investigation in our lab (Dr S. Ridewood, personal communication). Psta1, part of the Plasmodium sub-telomeric a family (Carlton et al., 2002;Templeton, 2009), was not annotated as essential on the piggyBac screen, but its disruption resulted in a strong fitness cost. Psta1 was chosen for genetic interrogation with the close paralogue Psta2 (PF3D7_1476800), which is most active at schizont stage and was annotated as essential.  Table S1.

OPEN ACCESS
In addition, we selected two PfSHs of unknown function that have also been annotated as essential: abH112 and abH114 (PF3D7_1143000). abH112 activity peaks in early schizont stage, and abH114 in early schizont and merozoite stages ( Figure 3B). Only abH114 has an ortholog in Plasmodium berghei, PBANKA_0906000, which was annotated as dispensable based on the PlasmoGEM genetic screen (Schwach et al., 2015). However, there are examples of differences in lipid metabolism pathways between rodent and human malarial parasites (Dé champs et al., 2010a(Dé champs et al., ,2010b. abH112 is conserved among humaninfecting parasites, and Psta1 and Psta2 are conserved in other Plasmodium laverania sub species, such as Plasmodium reichenowi and Plasmodium gaboni. Large-scale genetic screens are a very useful resource; they can be combined with complementary molecular biology techniques such as conditional KO (cKO) to validate gene essentiality.
cKO allows us to monitor the downstream effects of gene loss in real-time. To specifically determine whether the activity of selected SHs is essential, we designed a strategy to conditionally mutate the catalytic Ser to Ala which combines DiCre, artificial introns containing a LoxP site (LoxPint; Matera and Wang, 2014; Pieperhoff et al., 2014), and selection-linked integration (SLI). The DiCre system uses Cre recombinase constitutively expressed as two domains fused to rapamycin (RAP) binding domains. RAP treatment leads to dimerization and activation of DiCre and subsequent rapid excision of LoxP flanked DNA sequences (Collins et al., 2013a;Jullien et al., 2003). Our constructs contain a human dihydrofolate reductase (hdhfr) cassette to select for transfected parasites using the drug WR99210 (WR). In SLI, a neomycin phosphotransferase gene (npt) confers resistance to the drug G418. The npt is placed downstream of the modified gene of interest (GOI) via a viral ribosome skipping T2A peptide (Kim et al., 2011). After plasmid integration by single homologous recombination, the GOI and npt are translated as two proteins from a single mRNA, thus allowing us to select for integration (Birnbaum et al., 2017).
Our constructs (pT2A-cMUT) contain an N-terminal homology region (HR) to target the construct to the GOI. The rest of the recodonized gene (including the wild-type catalytic region, RR1), a C-terminal triple HA tag, the T2A coding sequence and npt are flanked with LoxPint sequences. This is followed by a second version of the recodonized region (RR2) containing an Ala codon in place of the catalytic Ser. RR2 is fused to gfp via a T2A peptide sequence. After transfection and selection of WR resistant parasites (1-2 weeks), G418 is added to select for parasites that have integrated the plasmid. RAP treatment of G418-resistant parasites induces excision of LoxPint flanked DNA sequences, resulting in loss of npt, replacement of the RR1 by RR2, and expression of a Ser to Ala SH mutant (SH-cMUT, Figure 4A) and free GFP.
Schizonts purified from a culture of P. falciparum DiCre-expressing B11 parasites (Perrin et al., 2018) were transfected with pT2A-cMUT constructs designed for each GOI ( Figure 4A). Four parasite lines were generated (Psta1-cMUT, Psta2-cMUT, abH112-cMUT and abH114-cMUT), and two clones selected for each one. Integration in pre-clonal and clonal lines was assessed by PCRs to detect the endogenous gene or 3' and 5' integration sites ( Figure 4A). Integration of the constructs into Psta2, abH112 and abH114 loci was highly . Genetic interrogation of P. falciparum SHs (A) Conditional allelic replacement strategy. The p2TA-cMUT construct has a homology region (HR, grey), loxPint sites (pink triangles), two differentially recodonized regions (RR1& RR2, turquoise), a triple HA tag (yellow), a T2A peptide sequence (purple), a npt selection marker (orange), gfp (green) and a hdhfr cassette (white). Upon RAP treatment, the sequence between the two loxPint sites is excised (post-excision locus). Arrows and red hexagons indicate start and stop codons, respectively. Primer binding sites are shown with dashed lines and half arrows. (B) Diagnostic integration PCRs. Primers were design to detect the endogenous locus or the 5' and 3' end integration sites. PCRs were performed on gDNA from pre-clonal (mix) and 2 clonal lines. B11 gDNA was used as a negative control. For abH112-cMUT and abH114-cMUT PCR conditions to observe 5' integration could not be obtained. ll OPEN ACCESS iScience Article efficient, as shown by the absence of an endogenous PCR product in the pre-clonal population ( Figure 4B). However, an endogenous PCR product was observed for pre-clonal Psta1 ( Figure 4B). 5 0 integration PCRs were never obtained for abH112 or abH114. Because all subsequent experiments supported integration, it was assumed that this was due to unyielding chromatin structure.
To test the excision efficiency of each cMUT line, synchronous parasite cultures were treated for 3h with DMSO or RAP at ring stage. Parasite samples were collected at schizont (Psta2-cMUT, abH112-cMUT, abH114-cMUT) or merozoite (abH112-cMUT) stages to extract genomic DNA (gNDA) and prepare protein lysates. As expected, RAP treatment resulted in smaller PCR product for Psta1, Psta2 and abH112, or a lack of it for abH114 ( Figure 4C). These results demonstrate efficient excision in all lines. Western blot (WB) analysis of parasite lysates confirmed the loss of the HA-tagged SHs after excision for all lines, except for one abH112 clone (5G) for which no HA signal was detected ( Figure 4D).
After validation of excision efficiency at the DNA and protein level, we looked at the effect of conditional mutation of the catalytic Ser on parasite replication. Synchronous ring-stage parasites from each cMUT line were treated with DMSO or RAP at ring stage. After 12h, RAP was removed, and cultures were transferred to 96-well plates (0.5 hematocrit at 0.1% parasitemia). Samples were collected every 48h, and parasitemia quantified by flow cytometry over 3 to 4 cycles ( Figure 4E). None of the allelic replacement mutants showed a significant decrease in parasite replication, indicating that the catalytic activities of Psta1, Psta2, abH112 and abH114 are not important for parasite development in culture. This is in contradiction to the large-scale genetic screening results (Zhang et al., 2018). However, we cannot rule out that these proteins might play an important non-catalytic function in P. falciparum.

Human SHs may be potential antimalarial targets
Upon RBC invasion, parasites remodel the RBC, and some host enzymes and pathways are co-opted. Exploring the interaction between parasite and host SHs could provide a new source of antimalarial targets. We identified 8 active HsSHs in iRBCs (Figures 3A and Table 2). Although saponin permeabilization should preclude the detection of HsSHs, some RBC membrane debris could remain in the parasite pellet. This could explain the presence of membrane associated SHs such as acetylcholine esterase (AChE, Ott, 1985). Alternatively, cytosolic HsSHs could be imported into the parasite as has been reported for other RBC metabolic enzymes such as ALAD and hPrx-2 (Bonday et al., 2000; Koncarevic et al., 2009). The RBC performs no de novo protein synthesis. Differences in HsSHs activity could reflect degradation of active enzyme, or a change in activation state or localization. All identified HsSHs showed some life-stage-dependent change in active enzyme abundance ( Figure 3A). Platelet activating factor acetyl hydrolase 1B gamma (PAFAH1B3) and acyl-amino acid releasing enzyme (APEH) were most activate at merozoite stage, the acyl protein thioesterases (LYPLA1 and LYPLA2) and fatty acid synthase (FASN) at ring and trophozoite stages, and AChE, neutral cholesterol esterase (NCEH1) and neuropathy target esterase (PNPLA6) at trophozoite and schizont stages. These results suggest that the enzymes may have stage-specific roles to play in parasite growth and replication. Many of these HsSHs have been extensively characterized, and inhibitors are commercially available (Table 2 and Figure 5A). We therefore purchased eight inhibitors to measure their antiparasitic activity.
Five of the inhibitors tested have been shown to be highly selective in mammalian systems using FP probebased ABPP studies: P11 selectively and reversibly inhibits PAFAH1B3 and the related PAFAH1B2, which was not identified in this study ; ML-348 and ML-349 selectively and reversibly inhibit LYPLA1 and LYPLA2, respectively (Adibekian et al., 2010a(Adibekian et al., , 2010b(Adibekian et al., , 2012; JW480 is a highly potent and selective reversible inhibitor of NCEH1 (Chang et al., 2011); and AA74-1 selectively inhibits APEH (Adibekian et al., 2011). TVB-3166 reversibly inhibits FASN via the keto-reductase unit (Huesca et al., 2005). This disrupts palmitate synthesis in vivo and on-target selectively was demonstrated by rescue with exogenous palmitate (Heuer et al., 2016;Ventura et al., 2015). TOCP is an organo-phosphophosphate (OP). OPs are responsible for human OP neuropathy, caused by inhibition of HsSHs in the brain. TOCP has been shown to be selective for PNPLA6 over AChE (Johnson, 1969(Johnson, , 1975Veronesi et al., 1991). The carbamate pyridostigmine bromide (PyBr) can protect from AChE-dependent OP poisoning by reversible inhibition of AChE and has been shown to target the erythrocyte variant (Dawson, 1994;Herkert et al., 2011;Koster, 1946  GPI anchored to the plasma membrane in erythrocytes (Ott, 1985). Possible nonesterase, membrane scaffolding activities (Soreq and Seidman, 2001). Nitric oxide signal transduction (Saldanha, 2017). iScience Article Ring-stage parasites were treated with DMSO or different concentrations of inhibitor for 72h, and parasitemia was measured by flow cytometry. The LYPLA1, APEH, PNPLA6, FASN, and AChE inhibitors were all found to affect parasite replication in a dose dependent manner, although full dose response curves were not obtained for FASN and AChE ( Figure 5B). The inhibitors of the other three HsSHs had no effect in parasite replication, including the LYPLA2 inhibitor ML349 ( Figure 5B). This was surprising because LYPLA1 and LYPLA2 have been shown to have the same biochemical function and share 60% identity (Long and Cravatt, 2011). These results support our hypothesis that some HsSHs may be co-opted by the parasite and are essential for growth and replication. We further interrogated the inhibition of APEH and LYPLA1 because their inhibitors showed the lowest EC 50 values and completely blocked parasite replication at high concentrations (Table 2 and Figure 5B).
To better understand the effect of LYPLA1 and APEH inhibitors in parasite development, we used a newly developed flow cytometry assay able to monitor the P. falciparum erythrocytic cycle with high temporal resolution (Bell et al., 2021). In this assay, fixed parasite cultures are stained with selective DNA (Hoechst) and RNA (132A) dyes (Cervantes et al., 2009). By monitoring the level of DNA and RNA in iRBCs we can track a synchronous parasite culture throughout the life cycle and distinguish between uRBCs, low background staining signals; rings, low DNA and RNA content; trophozoites, low DNA, medium RNA content; and schizonts, high DNA and RNA content ( Figure S2). Thus, using this assay we can determine the stage at which inhibitors arrest parasite development.
Synchronous parasites were treated immediately after RBC invasion (1 hpi) with 5 mM ML348 or 0.625 mM AA71-1 (3 3 EC 50 value, Table 2). DMSO was used as a negative control, and chloroquine (CQ) as a positive control that kills parasites immediately after treatment. Samples were collected and fixed at 24, 41, 48 and 72 hpi. Total parasitemia and the median DNA and RNA content of treatment cycle parasites was measured by flow cytometry ( Figure 5C).
Parasites treated with DMSO developed as expected, i.e. increase in RNA/DNA signals between 24 and 48 hpi and an increase in parasitemia reflected by a large population of rings with low RNA/DNA levels, whose RNA signal increases in trophozoite stage at 72 hpi ( Figures 5C and S2). None of the small molecules used affected uRBC morphology ( Figure S2). CQ-treated samples did not progress indicating ring-stage death ( Figures 5C and S2). ML348-treated parasites appeared to slow their development throughout the life cycle. At 48 hpi, iRBCs show significantly lower DNA and RNA levels compared to DMSO controls (Figure 5C). Parasitemia increased slightly, indicating that ML348 significantly delays schizogony but a small proportion of parasites are able to progress to the next cycle at 5 mM ML348 ( Figure S2). Our ABPP results indicated that LYPLA1 is most active at ring and trophozoite stages ( Figure 3A). Taken together, these data suggest that if LYPLA1 is the main target of ML438, its metabolic function in trophozoites is likely important for parasite to successfully undergo schizogony.
AA74-1-treated parasites appeared to arrest development early in the cycle similarly to CQ-treated ones ( Figure 5C). Indeed, no significant increase in DNA and RNA levels was observed between the 24 hpi and the later time points. This also indicates that AA74-1 is a very fast acting compound. Our ABPP results indicate that APEH is most active in merozoites ( Figure 3A), suggesting that it is internalized in parasites and that it has a role immediately after invasion. iScience Article In order to assess the selectivity of the HsSH inhibitors, we tested them in competition with FP-TMR in parasite lysates. In schizonts, pre-treatment with AA74-1 inhibited FP-TMR labeling of two bands corresponding to full-length and processed APEH as previously described ( Figures 5D and 5E, Elahi et al., 2019a). When schizonts were saponin-treated before the competition assay, only the lower MW band was observed, suggesting that only the processed form of this protein is internalized in the parasite. These data support the findings of a recent article that showed evidence for APEH internalization and its essential role in P. falciparum asexual replication (Elahi et al., 2019). In ring lysates, competition of the reversible inhibitor ML348 with sub-saturating concentration of FP-TMR prevented labeling of a single band at the expected molecular weight for LYPLA1 (25kDa, Figure 5D). A band was observed at the same MW by WB, but there was too much non-specific binding to be confident that this is LYPLA1 ( Figure 5E). Although these data point to the specificity of AA74-1 and ML348 for APEH and LYPLA1 respectively, there may be low abundant or lowly/ unlabeled targets that are not identified by competition ABPP. To test if PfSH targets are involved in AA74-1 or ML348-dependent growth inhibition, a chemical proteomics strategy could be employed similar to techniques recently used to find targets of Salinipostin A (Yoo et al., 2020).

DISCUSSION
In recent years, the need for new antimalarial treatments with novel modes of action has led to a refocus on target-based drug discovery in the field. Metabolic SHs play essential functions in many biochemical processes, such as lipid metabolism and posttranslational modification. SHs have been proven to be druggable, but they remain unexplored as a source of potential antimalarial targets. In this study, we have used the cell-permeable FP-N 3 probe conjugated to biotin via CuAAC-click chemistry and TMT-based isobaric labeling to provide the most in-depth study of Plasmodium SHs and quantify changes in their activity throughout the erythrocytic cycle in intact parasite.
In our study, we profiled the life-stage specific activity of 25 PfSHs and 8 HsSHs. Among the PfSHs, we identified 22 out of 43 predicted a/bHs (Table 1), and 3 out of 4 predicted PLPs. SHs that are exported into the RBC/PV lumen were expected to be lost after saponin permeabilization. However, the identification of PfEH1 and PfXL2, which contain the Plasmodium PEXEL export element motifs (Hiller et al., 2004;Marti et al., 2004), suggests that some exported hydrolases are still detectable using our methods, especially if they are associated with the RBC membrane. Indeed, PfEH1 has been shown to localize at the periphery of iRBCs and is associated with spectrin (Spillman et al., 2016). The remaining PfSHs may not be expressed or might be below the detection limit. Alternatively, they may be present in an inactive form or within organelles inaccessible to FP-N 3 . Finally, FP ABPs have been shown to target only 80-90% of metabolic SHs in complex biological samples (Kidd et al., 2001), and some of the predicted PfSHs might be pseudoenzymes (Zimmermann, 2013).
The majority of PfSHs identified are unannotated, and for many, we have reported the first evidence of a serine nucleophile in their active site. All PfSHs showed some change in activation during the erythrocytic life cycle. This suggests time-dependent activation that could be linked to the distinct metabolic roles and requirements of the different asexual life stages. The activation heat map presented here (Figure 3) is likely to be a useful tool in any further investigation into this large and relatively uncharacterized enzyme family. Of interest, some PfSHs demonstrate a significant increase in activity levels between mature schizont and merozoite stages, for example Psta1 and RhoSH. Merozoite egress is a rapid and tightly regulated process, thus activity increases around this event are unlikely to be due to changes in protein expression or translation but rather specific activation. ABPP is well suited to detect these changes. RhoSH has recently been studied in our lab using conditional genetics. Conditional mutation of the catalytic triad His to Ala causes a defect in PVM formation immediately after invasion that results in a significant delay in intracellular development and a decrease in parasite replication (Dr S. Ridewood, personal communication). This is consistent with the subcellular localization of RhoSH in the rhoptries (apical secretory organelles that are important for invasion and PVM formation). The proposed function of RhoSH in PVM formation immediately after invasion is supported by the merozoite-specific activation shown here by ABPP.
In this study, four PfSHs were selected for genetic validation in P. falciparum: abH112, abH114, Psta1 and Psta2. These candidates were prioritized based on their annotated essentiality or importance in highthroughput genetic screens (Zhang et al., 2018). Surprisingly, conditional mutation of their catalytic Ser showed no change in parasite replication. However, our conditional mutation results cannot rule out essential non-catalytic functions for these proteins. These PfSHs could also have an essential role in lipid ll OPEN ACCESS iScience 25, 104996, September 16, 2022 15 iScience Article metabolism in vivo that is not observable in cell culture, where parasites have an abundance of lipids and other nutrients in the media. Genes can be miss-annotated as essential in large scale genetic screens due to competition effects between co-cultured mutant lines. This has been reported before for other genes annotated as essential in the piggyBac screen such as the merozoite surface protein 1 (MSP1) or dipeptidyl aminopeptidase 3 (DPAP3) whose cKO only results in a 50% decrease in parasite replication (Das et al., 2015;Lehmann et al., 2018). By combining the wealth of data from large-scale genetic screening annotations with more targeted molecular biology approaches we can powerfully increase the quality of genome annotation.
One of the most interesting results from our ABPP study was the identification of 8 active HsSHs in iRBCs. We demonstrate that inhibition of APEH, LYPLA1, FASN, PNPLA6 or AChE blocks parasite replication in vitro, with the APEH inhibitor AA74-1 and the LYPLA1 inhibitor ML348 being the most potent. From our ABPP results, LYPLA1, a predicted depalmitoylase, seems to be most active at ring and trophozoite stages. However, its inhibition only had an effect later in the cycle where a significant delay in schizogony and a block in egress was observed. P. falciparum employs palmitoylation machinery both in the parasite and RBC lumen, possibly both parasite and host derived (Kilian et al., 2020). LYPLA1 may be involved in this machinery. S-palmitoylated proteins have been shown to be abundant in the late trophozoite and schizont stages and essential for merozoite development (Jones et al., 2012;Kilian et al., 2020). Depalmitoylases in ring and trophozoite stage could be important in recycling palmitate ready for the later phases of asexual development. APEH is most active in merozoites, suggesting that it might be imported into the parasite and that it might play a role in invasion or early ring development. Interestingly, the APEH inhibitor displays sub-micromolar anti-parasitic activity and quickly kills parasites at ring stage. A recent article confirmed our hypothesis about the essentiality of APEH in asexual-blood stages and its import into the parasite (Elahi et al., 2019a). The endopeptidase activity of APEH, which cleaves N-acetylated amino acids from peptides, was suggested to play a role in the degradation of erythrocytic proteins in the parasite food vacuole (Elahi et al., 2019a).
These HsSHs represent an exciting new area of research to develop anti-parasitic drugs. A number of other human proteins are thought to be co-opted by the parasite to perform functions such as haem biosynthesis (Bonday et al., 1997;Nagaraj et al., 2013;van Dooren et al., 2012). Host enzymes are attractive targets as they will not accumulate drug-resistance mutations. However, other mechanisms of resistance may arise, such as enzymatic inactivation of the drug or active efflux. Many human metabolic SHs have been pursued as promising drug targets for human diseases, such as AChE for Alzheimer's disease (Saxena and Dubey, 2019). This opens the possibility of repurposing HsSH inhibitors as potential antimalarial drugs, although the toxicity of these inhibitors in humans has not been assessed. LYPLA1 inhibitor ML348 has no cytotoxicity effect in human embryonic kidney cells (HEK293T) up to 50mM (Adibekian et al., 2010a). Although AA74-1 has been shown to be highly selective for APEH in mice (4h treatment at 0.2-1.6mgkg À1 , intraperitoneal) mammalian toxicology studies have not been done (Adibekian et al., 2011). Finally, if the co-opted function of the host enzyme is essential for all malaria-causing Plasmodium species, the antimalarial drug should be equally effective as a treatment for all, as opposed to a drug that target a conserved parasite enzyme that might show different potencies among Plasmodium homologues. The targeting of human enzymes to fight infectious diseases has shown promise in virology. For example, human N-myristoyltransferases are coopted for viron assembly, and their inhibition blocks virus replication and infectivity (Mousnier et al., 2018). This has been demonstrated in highly diverse viruses like Rhinovirus, responsible for the common cold, whose high replication and mutational rate have hampered any efforts to develop vaccines (Mousnier et al., 2018;Thibaut et al., 2012). Overall, targeting the host enzyme is a promising new avenue in the development of treatments against infective agents that rapidly acquire resistance to pathogen-directed drugs.
The study of human and Plasmodium metabolic SHs is a highly interesting field. Many PfSHs are predicted to be lysophospholipases or lipases (Table 1), and therefore may play important roles in lipid and phospholipid scavenging, processing or synthesis. The phospholipid content of RBCs increases almost five-fold during intra-erythrocytic parasite growth, and the source of PVM lipids is still a topic of debate (Sherling and van Ooij, 2016;Tran et al., 2016). Lipids also play significant roles in signaling and immunogenic functions (Jiang et al., 2012). Mature erythrocytes do not perform de novo fatty acid synthesis because they lack acetyl-CoA carboxylase which provides the type 1 human fatty acid synthase (FASN) with malonyl-CoA (Pittman and Martin, 1966). The Plasmodiumtype II FASN pathway has been shown to be non-essential to asexual-blood stage replication (Vaughan et al., 2009). Therefore, the parasite may co-opt human ll OPEN ACCESS iScience 25, 104996, September 16, 2022 iScience Article proteins to create a full fatty acid synthesis network in iRBCs. ABPP, metabolic chemical proteomics, lipidomics and metabolomics all have important roles to play in deciphering these complex biological processes and the interplay between host and parasite enzymes.
Genome annotation is of great importance in Plasmodium where only two thirds of the genes have any predicted function, few of which have been experimentally validated (Bö hme et al., 2019). This study illustrates the benefits of ABPP-chemical proteomics to functionally annotate the genome based on a common catalytic mechanism. The distinction between active and non-active enzyme forms helps to align enzyme activity with life-stage specific functions, for instance the highly regulated events around merozoite egress. SH and protease ABPs were among the first to be described and certainly the most widely used (Greenbaum et al., 2000;Liu et al., 1999). Probes have also been developed towards glycosidases (Vocadlo and Bertozzi, 2004;Wu et al., 2019), phosphatases (Kalesh et al., 2010) and PLP-dependent enzymes (Hoegl et al., 2018). After the success of this in-depth ABPP study of SHs, we propose that ABPP should play a central role in the functional annotation of the Plasmodium genome.

Limitations of the study
A limitation of the LCMS-MS method is that lowly abundant proteins may be masked by larger peaks from highly abundant proteins, therefore causing us to miss some predicted PfSHs. Ratio compression is also a problem when using isobaric labeling but we have attempted to minimize this by using fractionation to reduce sample complexity. We have used the soluble fraction of parasite lysates and despite the use of Triton-X100 there may be SHs in the insoluble fraction that have been missed. The reaction of a predicted SH with the FP probe does not necessarily mean that the catalytic activity of the SH is related or important to its main cellar role. This may be why the predicted essential PfSHs we interrogated did not show any essentiality in our hands. There may also be limitations to our experimental model showing different essentialities than would emerge under less nutrient-rich conditions or in vivo.
When considering the activity changes of the HsSHs, there is a limitation inherent in sample collection as there are many more human proteins present in ring stage-than in schizont stage-infected erythrocytes. Finally, although the HsSH inhibitors that we have used all have demonstrated specificity in mammalian systems they could still have P. falciparum off-targets.

LCMS/MS data analysis
The raw data files were analyzed using MaxQuant version 1.6.0.1 (Tyanova et al., 2016a). Quantification was done at MS2 level using TMT-six/tenplex labels. All other MaxQuant settings were kept the same as default. The MaxQuant search engine Andromeda (Cox et al., 2011) was used with sequence databases Homo sapiens (Uniprot 13/01/2013) and Plasmodium falciparum 3D7 (PlasmoDB 15/12/2016). A decoy database of reversed sequences was used to filter false positives at a peptide false detection rate of 1%.
The data files generated by MaxQuant were further analyzed using Perseus version 1.5.6.0 (Tyanova et al., 2016b). The list of identified proteins was first filtered to remove potential contaminants, proteins only identified by one site, and proteins identified from the reverse (decoy) peptides. The protein intensity values were then transformed by function log 2 (x). The log 2 intensities were normalized by subtracting the median intensities of each replicate followed by the median intensities of each protein. Proteins identified from fewer than 4 peptides were filtered out at this stage. Student's t-tests were performed to identify proteins with statistically significant changes between conditions using stringency parameters s0 = 0.5 and FDR = 0.01.

Generation of conditional mutant lines
The constructs for the conditional mutation of the catalytic Ser of selected PfSHs ( Figure 4A) were based on the pT2A_PfDdi1_cWT construct (kind gift of Dr S. Ridewood, which was in turn based on the pT2A_-FIKK10_cKO construct, kind gift of Dr M. Treeck, The Francis Crick Institute). The plasmid backbone contains a T2A and gfp sequence followed by a P. berghei dihydrofolate reductase thymidylate synthase for expression of the GOI, and a hdhfr cassette to select for transfected parasites with WR99210. The pT2A_-GOI-cMUT plasmids were obtained by combining four DNA fragments via in-fusion PCR using Phusion High-Fidelity DNA Polymerase (New England Biolabs) and the In-Fusion HD Cloning Kit (Clontech) as per manufacturer's instructions. The four DNA fragments containing 15 nucleotides overlapping sequences were: First, the plasmid backbone obtained after digestion of pT2A_PfDdi1_cWT with SalI and BglII (New England Bioscience). Second, the HR upstream of the catalytic Ser that was amplified from P. falciparum B11 gDNA purified using a DNeasy Blood and Tissue Kit (Qiagen). The third and fourth fragments were PCR amplified from synthetic DNA (GENEWIZ). The third includes the first loxPint, the RR1 and the triple HA tag, and the fourth the npt gene, the second loxPint and the RR2 containing the catalytic Ser to Ala mutation. RR1 was recodonized for Escherichia coli and RR2 for Oncorhynchus mykiss. Oligonucleotide primers were used at 200 nM (Table S1). PCR and digestion products were purified using QIAquick PCR Purification Kit (Qiagen) as per manufacturer's instructions. Oligonucleotide primers were obtained from Sigma. PCRs were carried out using a thermocycler, and amplicons were analyzed by examining 1% agarose gel electrophoresis stained with SYBR Safe (Thermo Fisher Scientific). DNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). pT2A_GOI-cMUT plasmids were amplify in XL10-Gold Ultracompetent cells (Aligent) and purified using QIAprep Midi Kits (Qiagen). Plasmids sequences were checked via capillary sequencing by Source BioScience.
Plasmids were transfected into P. falciparum B11 using an Amaxa 4-D electroporator (Lonza) as follows. 10 mg of pT2A_GOI-cMUT plasmid were ethanol precipitated, re-suspended in 10 mL sterile TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and mixed with 15-20 mL of Percoll-purified mature schizonts suspended in 100 mL of Amaxa Primary Cell solution P3 (P3 Primary cell 4-D Nucleofector X kit, Lonza). This mixture was electroporated using the FP158 pre-set condition, then immediately transferred to 2 mL of RBCs suspended in complete media (20% hematocrit) and incubated for 30 min at 37 C with shaking to allow egress and invasion to take place. A further 8 mL of complete media was then added, and the cultures were incubated at 37 C under standard conditions overnight. The media was then replaced and supplemented with 2.5 nM WR99210 (WR, Jacobus Pharmaceuticals) to select for transfected parasites. When WR resistant parasites were observed at 1% parasitemia (approximately 2 weeks), Geneticin Selective Antibiotic G418 Sulphate (G418, Gibco) was added at 450 mL/mL to select for chromosomal integration of the plasmid. When 1% parasitemia of WR and G418 resistant parasites was observed (2-4 weeks), samples were taken for cryopreservation, and gDNA extracted to confirm integration by PCR. Clonal lines were obtain using a plaque assay: cultures at 0.75% hematocrit were serially diluted in 96-well flat-bottom plates (Corning Costar) and cultured for 12 days in media containing WR and G418. Plates were then inspected using an inverted light microscope to identify wells containing a single plaque corresponding to a single clonal parasite population. The content of some of these wells (at least 5 per cMUT line) were expanded, and integration verified by PCR. Excision efficiency at the DNA level was measured by diagnostic PCR. Synchronous

OPEN ACCESS
To determine parasitemia by flow cytometry, fixed samples were stained with 1 mg/mL of Hoechst 33342 (Thermo Fisher Scientific) in PBSa for 30 min at 37 C. Five to ten thousand RBCs per samples were analyzed by flow cytometry using a high-throughput Fortessa Analyzer (BD Biosciences) and the FACSDiva Software v8.0.1. Infected RBCs were distinguished from uRBCs as the population that showed positive DNA staining using a 355 nm UV excitation laser and a 450/50 nm emission filter. Flow cytometry data were analyzed using FlowJo LLC 2006-2015. For inhibitor treatment, EC 50 values were obtained by fitting the parasitemia to a dose response curve in Prism.

Phenotypic characterization of HsSHs inhibitor treatment by flow cytometry
To determine which stage of parasite development was affected by the LYPLA1 and APEH inhibitors ML348 and AA74-1, we used a recently developed flow cytometry assay that allows the dissection of the erythrocytic cycle with hourly resolution. This assay uses highly specific DNA (Hoechst) and RNA (132A, kind gift of Prof Y.T. Chang, National University of Singapore) dyes to monitor intracellular parasite development. Although DNA signal in iRBCs only increases during schizogony because of DNA replication, RNA levels increase throughout the cycle, thus allowing us to monitor the ring to trophozoite transition. In addition, this assay also allows us to monitor egress and invasion given the big difference in DNA and RNA content between mature schizonts and rings.
A synchronous culture of P. falciparum 3D7 parasites at 2% parasitemia was treated at ring stage with 3 times the EC 50 values of ML348 (5 mM) or AA74-1 (0.625 mM). DMSO and CQ (2 mM) were used as negative and positive controls. Samples were collected 24, 41, 48, and 72 h after treatment, fixed and stored as described above, stained for 30 min with 1 mg/mL Hoechst and 2 mM 132A at 37 C, and analyzed by flow cytometry. 132A signal was measured using a 488nm excitation laser and a 610/20 nm emission filter. DNA and RNA levels in iRBCs were calculated as the median DNA and RNA signal of iRBCs divided by that of uRBCs. For those time points in which two distinct populations of iRBCs were observed, generally schizont and ring stage parasites, the DNA and RNA content of these two populations were calculated independently because these populations correspond to iRBCs belong to the first or second cycle after treatment.

OPEN ACCESS
iScience 25, 104996, September 16, 2022 29 iScience Article QUANTIFICATION AND STATISTICAL ANALYSIS Growth assay quantification was represented by the mean G standard deviation as indicated in figure legends and calculations were performed using GraphPad Prism Software. Quantification and statistical analysis of proteomic data was done using MaxQuant version 1.6.2.1 (Tyanova et al., 2016a) and Perseus version 1.5.6.0 (Tyanova et al., 2016b) as described in the LCMS/MS data analysis section of the STAR Methods. The statistical tests used are one-way ANOVA and Student's t-test, as indicated. p % 0.001, ***; p % 0.01, **; p % 0.05, *; p < 0.05, non-significant.